This invention is generally in the area of antibodies to plasma proteins, specifically Protein C, and methods for use thereof.
Protein C is a vitamin K-dependent plasma protein zymogen to a serine protease. Upon activation it becomes a potent anticoagulant. Activated protein C acts through the specific proteolysis of the procoagulant cofactors, Factor VIIIa and Factor Va. This activity requires the presence of another vitamin K-dependent protein, protein S, calcium and a phospholipid (presumably cellular) surface. Referring to FIG. 1, from Hemostasis and Thrombosis: Basic Principles and Clinical Practice 2nd Ed., Colman, R. W., et al., p. 236(J. B. Lippincott, Philadelphia, Pa. 1987), protein C circulates in a two-chain form, with the larger, heavy chain bound to the smaller light chain through a single disulfide link. A small proportion of the protein also circulates in a single chain form, where a lys-arg dipeptide in the molecule connects the light chain directly to the heavy chain. Protein C is activated to activated protein C (APC). Thrombin is capable of activating protein C by the specific cleavage of the Arg.sup.12 -Leu.sup.13 bond in the heavy chain. In vivo, in the presence of physiological concentrations of calcium, the rate of this activation is enhanced dramatically when thrombin is bound to the endothelial cell cofactor, thrombomodulin. Matschiner, et al., Current Advances in Vitamin K Research, pp. 135-140, John W. Suttie, ed. (Elsevier Science Publishing Co., Inc. 1988) have further reviewed the role of the Vitamin K dependent proteins in coagulation.
Protein C has been shown to have major importance in vivo. Patients deficient in protein C, or its cofactor, protein S, show pronounced thrombotic tendencies. Babies born totally deficient in protein C exhibit massive disseminated intravascular coagulation (DIC) and a necrotic syndrome which leads to death within the first few weeks of life if untreated. Activated protein C has also been shown to protect animals against the coagulopathic and lethal effects of endotoxin shock, as described by Taylor, et al., in J. Clin. Invest. 79, 918-925 (1987).
As first reported by Kisiel, in J. Clin. Invest. 64, 761-769 (1979), Protein C was originally isolated in semi-pure form from plasma using classic protein purification techniques, including barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. This procedure was vastly improved and facilitated by the discovery of a unique antibody to Protein C, designated HPC-4, described by Stearns, et al., in J. Biol Chem. 263(2), 826-832 (1988). As detailed by Esmon, et al., at the Joint IABS/CSL Symposium on Standardization in Blood Fractionation including Coagulation Factors, Melbourne, Australia 1986 (reported in Develop. biol. Standard., 67, 51-57 (S. Karger, Basel, 1987), Protein C can be isolated from human plasma by batch adsorption of diluted heparinized plasma on QAE Sephadex, washing with buffered 0.15M NaCl and eluting with 0.5M NaCl, recalcifying and batch adsorbing with HPC-4, then washing with a Ca.sup.2+ containing buffer and eluting the Protein C with an EDTA containing buffer.
HPC-4 is a calcium-dependent monoclonal antibody to human protein C. The epitope recognized by the antibody has been identified and corresponds to the stretch of amino acids in the zymogen of protein C which spans the thrombin cleavage site. Activated protein C is not recognized by HPC-4.
Several antibodies to human protein C have been reported, for example, by Laurell, et al., FEBS Letts. 191(1), 75-81 (1985); Wakabayashi, et al., J. Biol. Chem. 261, 11097-11105 (1986); Sugo, et al., Thromb. Hemost. Abstrs., Brussells, 229 (1987); and Ohlin, et al., J. Biol. Chem. 262, 13798-13804 (1988). Some of these are calcium dependent, for example, one of the antibodies reported by Laurell, et al. However, as far as can be determined in the published reports, this dependence is due to the requirement for calcium binding to the light chain of protein C and the antibodies recognize epitopes on the light chain. Other antibodies recognize the region around the thrombin cleavage site on the heavy chain, but these are not calcium dependent, including the HPC-4 described by Ohlin, et al. The HPC-4 antibody of Ohlin, et al., is not both Ca.sup.2+ dependent and directed against the activation region, and is therefore different from the antibody of the present invention.
None of the other antibodies that bind to the Ca.sup.2+ binding region of Protein C recognize only Protein C and not the activated form, Situations may arise in which the protein uncontaminated by its active form is desirable. This is particularly the case with reference to therapeutic uses of the antibody to inhibit Protein C activation.
Unfortunately, although the use of the antibody of the present invention, and, more recently, the properties, have been reported in the literature, it has not been publicly available for use in isolation, diagnostic, or therapeutic methodologies.
It is therefore an object of the present invention to provide a Ca.sup.2+ dependent antibody which binds to the activation region of Protein C.
It is a further object of the present invention to provide a method and means for using a domain of this Ca.sup.2+ dependent antibody for isolation of other non-metal binding peptides or proteins by metal dependent affinity chromatography.
It is a still further object of the present invention to provide a method and means for using this Ca.sup.2+ dependent antibody for therapeutic purposes.
It is yet another object of the present invention to provide this Ca.sup.2+ dependent antibody, antibodies, derivatives and conjugates thereof, for diagnostic purposes.